A fascinating diversity of molecular mechanisms has been found for the receptors of steroid hormones, including the adrenal glucocorticoids, which regulate many vital metabolic, developmental, immune, and inflammatory processes. In the classical model of steroid action, glucocorticoids bind to the glucocorticoid receptor (GR) in target cells. GR then interacts as a homodimer with DNA sites in the nucleus and induces transcription of nearby genes. We have discovered a fundamentally different mode of action in which a GR monomer heterodimerizes with a second DNA-binding protein. Although mechanisms involving tethered ternary complexes as well known, this is the first description, to our knowledge, of any member of the nuclear receptor superfamily forming a heterodimer with an unrelated protein, with both partners bound to the DNA. This mechanism controls activation of the gene for the gamma subunit of fibrinogen, the major blood-clotting protein, in Xenopus frogs. The heterodimerization partner, Xenopus glucocorticoid receptor accessory factor (XGRAF), binds to a DNA sequence not previously known as a protein binding site. Preliminary data suggest that XGRAF belongs to the basic helix-loop-helix class of transcription factors. The overall goal of this research is to understand how GR and XGRAF together activate transcription in response to hormone. The experimental strategy is to answer three basic questions: A) What is the identity of XGRAF? B) How does GR physically interact with XGRAF? C) What is the full complement of DNA: protein interactions underlying the action of XGRAF and GR? These questions will be addressed by the following specific aims: 1) Identify XGRAF by powerful methods of mass spectrometry and proteomics. 2) Isolate XGRAF cDNA clones. 3) Identify specific amino acids in GR required for XGRAF:GR cooperative binding to DNA. 4) Determine whether GR and XGRAF heterodimerizes in the absence of DNA. 5) Identify all nucleotides flanking the core GR and XGRAF binding sites that are critical for induction of transcription. This mechanism is of fundamental significance to the field of hormone action since it reveals new pathways by which a steroid response can be regulated by nonreceptor proteins. Furthermore, because receptor structure is highly conserved in the region of GR that interacts with XGRAF, it is likely that similar modes of regulation apply to GR, as well as other nuclear receptors, in other animals. This research is of significance to health and disease because glucocorticoids are widely-used for many common immune inflammatory disorders such as arthritis, asthma, and allergies, but the treatments are hampered by serious effects since these steroids act in virtually all cells. Targeted therapies are needed, and our work shows that specialized regulatory elements for particular genes could provide a way to modulate selectively the expression of some hormone-inducible genes, while leaving other unaffected.